Proteomic Analysis of Irradiation with Millimeter Waves on Soybean Growth under Flooding Conditions

Abstract

Improving soybean growth and tolerance under environmental stress is crucial for sustainable development. Millimeter waves are a radio-frequency band with a wavelength range of 1-10 mm that has dynamic effects on organisms. To investigate the potential effects of millimeter-waves irradiation on soybean seedlings, morphological and proteomic analyses were performed. Millimeter-waves irradiation improved the growth of roots/hypocotyl and the tolerance of soybean to flooding stress. Proteomic analysis indicated that the irradiated soybean seedlings recovered under oxidative stress during growth, whereas proteins related to glycolysis and ascorbate/glutathione metabolism were not affected. Immunoblot analysis confirmed the promotive effect of millimeter waves to glycolysis- and redox-related pathways under flooding conditions. Sugar metabolism was suppressed under flooding in unirradiated soybean seedlings, whereas it was activated in the irradiated ones, especially trehalose synthesis. These results suggest that millimeter-waves irradiation on soybean seeds promotes the recovery of soybean seedlings under oxidative stress, which positively regulates soybean growth through the regulation of glycolysis and redox related pathways.

Keywords: crop productivity; early-stage soybean; glycolysis; redox; seed irradiation.

Figure 1

Morphological effect of irradiation of millimeter waves on soybean. Soybean seeds irradiated with millimeter waves were sowed and treated, which is indicated in materials and method. Bar indicates 1 cm. Column graph shows the length of main root/hypocotyl and line graph shows the weight of main root/hypocotyl. Data are shown as means ± SD from three independent biological replicates. The different letters indicate significant changes according to one-way ANOVA followed by Tukey’s multiple comparisons (p < 0.05).

Figure 2

Functional categories of proteins in root-hypocotyl tissue with differential abundance in soybean treated with millimeter-waves irradiation and flooding. Soybean seeds irradiated with millimeter waves were sowed and treated, which is indicated in materials and method. Functional categories of significantly changed proteins (p < 0.05) from unirradiated/irradiated and unflooded/flooded during growth were determined using MapMan bin codes. Abbreviation: mitoETC, mitochondrial electron transport chain. “others*” contains proteins related to gluco-neogenesis/glyoxylate cycle, polyamine metabolism, S-assimilation, biodegradation of xenobiotics, N-metabolism, tetrapyrrole synthesis, C1-metabolism, Co-factor and vitamin metabolism, metal handling, and oxidative pentose phosphate pathway. “others**” contains proteins related to gluco-neogenesis, S-assimilation, transporter, polyamine metabolism, biodegradation of xenobiotics, tetrapyrrole synthesis, Co-factor and vitamin metabolism, oxidative pentose phosphate pathway, C1-metabolism, N-metabolism, metal handling, and fermentation. “not assigned” indicates proteins without ontology or characterized functions.

Figure 3

Mapping on metabolic pathways of differentially changed proteins identified from soybean seedling irradiated without (control) or with millimeter waves (treated) under flooded or unflooded conditions. (A) Mapping from unirradiated soybean under unflooded conditions. (B) Mapping from irradiated soybean under unflooded conditions. (C) Mapping from unirradiated soybean under flooding. (D) Mapping from irradiated soybean under flooding. Changed proteins were submitted to the MapMan software and mapped to metabolic pathways. Each square indicates one mapped protein. Color indicates the fold change value of a differentially changed protein. Red and blue colors indicate an increase and decrease in fold change values, respectively. Abbreviations: CHO, carbohydrate metabolism; MitoETC, mitochondrial electron transport chain; misc, miscellaneous; OPP, oxidative pentose phosphate pathway; TCA, tricarboxylic acid cycle.

Figure 4

Mapping of differentially changed proteins that related to glycolysis identified from root-hypocotyl tissue of irradiated (treated)/unirradiated soybean (control) during early seedling growth. Proteins were extracted from flooded and unflooded soybean seedlings. (A) Mapping from unirradiated soybean under unflooded conditions. (B) Mapping from irradiated soybean under unflooded conditions. (C) Mapping from unirradiated soybean under flooding. (D) Mapping from irradiated soybean under flooding. Proteins neither detected nor changed are marked in white. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase. The number in the box indicates the enzyme commission number of corresponding proteins.

Figure 5

Mapping of differentially changed proteins that related to ascorbate and glutathione metabolism identified from irradiated (treated)/unirradiated (control) soybean root. Proteins were extracted from flooded and unflooded soybean seedlings. (A) Mapping from unirradiated soybean under unflooded conditions. (B) Mapping from irradiated soybean under unflooded conditions. (C) Mapping from unirradiated soybean under flooding. (D) Mapping from irradiated soybean under flooding. Proteins neither detected nor changed are marked in white. The number in the box indicates the enzyme commission number of corresponding proteins.

Figure 6

Mapping of differentially changed proteins in soybean root related to sugar metabolism. Proteins were identified from soybean seedlings treated without or with millimeter-waves irradiation under flooded and unflooded conditions. Pathway map was determined on KEGG database. (A) Mapping from unirradiated soybean under unflooded conditions. (B) Mapping from irradiated soybean under unflooded conditions. (C) Mapping from unirradiated soybean under flooding. (D) Mapping from irradiated soybean under flooding. Proteins neither detected nor changed are marked in white. The number in the box indicates the enzyme commission number of corresponding proteins.

Figure 7

Immunoblot analysis of proteins involved in ascorbate/glutathione pathway. Proteins were extracted from soybean seedlings and separated on 10% SDS-polyacrylamide gel by electrophoresis and transferred onto membranes. The membranes were cross-reacted with anti-APX, anti-GR, and anti-PRX antibodies. CBB staining pattern was used as loading control (Figure S5). The integrated densities of bands were calculated using ImageJ software. Picture shows three independent biological replicates (Figure S6). Data are shown as the means ± SD from three independent biological replicates.

Figure 8

Immunoblot analysis of proteins involved in glycolysis pathway. Proteins were extracted and separated on 10% SDS-polyacrylamide gel by electrophoresis and transferred onto membranes. The membranes were cross-reacted with anti-FBPA, anti-TPI, and anti-GAPDH antibodies. CBB staining pattern were used as loading control (Figure S5). The integrated densities of bands were calculated using ImageJ software. Pictures shows three independent biological replicates (Figure S7). Data are shown as means ± SD from three independent biological replicates.